More than 300 rare genetic bone diseases have been identified but treatment for these disorders is usually limited because little of their pathogeneses is known. Current methods for studying these disorders include animal models or generating bone cells from patient blood or bone marrow. However, collecting specimens repeatedly from patients is often impractical and may be unethical, while animal models often fail to replicate the complete features of those diseases. Recent advance in patient-specific induced pluripotent stem (iPS) cell biology opened new avenues for studying bone cells from patients. In this 5-year proposal, the applicant plans to use patient-specific iPS cells to study craniometaphyseal dysplasia (CMD) with a focus on osteoclasts (OCs), the bone resorbing cells. The onset of CMD begins in childhood with thickening of craniofacial bones and abnormal shape of long bones. Its lifelong progression leads to life-threatening consequences in some patients. To date, there is no treatment other than repetitive surgery. Previous studies in a knock-in (KI) mouse model carrying a CMD-causing Ank mutation revealed OC defects in AnkKI/KI mice. Similar results were found in human peripheral blood cultures of CMD patients. AnkKI/KI OCs also showed slower movement with abnormal actin organization. To test the hypothesis generated from the CMD mouse model that CMD-causing ANK mutations reduce individual osteoclast activity by negatively affecting the actin cytoskeleton, four specific aims are proposed. In Aims 1 and 2 (K99 phase), the applicant will optimize the methods 1) for generating iPS cells from fibroblasts or SHEDs (stem cells from human exfoliated deciduous teeth) of control individuals and CMD patients and 2) for differentiating iPS cells into OCs. In Aim 3 (R00 phase), the applicant will compare iPS- derived OCs from healthy controls and CMD patients to identify differences in OC formation, function, expression of OC marker genes, adhesion and migration by TRAP staining, resorption pit assays, real-time PCR, adhesion assays and live-cell time-lapse imaging, respectively. In Aim 4 (R00 phase), the applicant will study the organization and dynamics of the actin cytoskeleton as well as the colocalization of ANK and actin in control and CMD OCs by confocal microscopy. Two critical regulatory mechanisms in actin biology, the activation of GTPase family members, Rac, Rho and Cdc42, and tyrosine phosphorylation in iPS-derived OCs of controls and CMD patients will be examined by active GTPase pull-down assays, immunostaining and immunoblots. The ultimate goal of this study is to use CMD as paradigm to establish novel tools for studying rare genetic skeletal disorders. Stem cell and bone biology are areas of major interest at School of Dental Medicine at UCHC. The research environment is exquisitely suited to perform this project and to develop the applicant's career in the Dentist/Scientist track. PUBLIC HEALTH RELEVANCE: This application uses human induced pluripotent stem (iPS) cells to study osteoclast defects in craniometaphyseal dysplasia (CMD), a rare genetic bone disorder. This study has the potential to reveal novel therapeutic targets for CMD treatment and to improve our understanding of osteoclast biology, which may benefit research in other rare genetic skeletal disorders and some common osteoclast-related disorders, such as osteoporosis or osteopetrosis.